Amyloids are filamentous polymers of aberrantly folded proteins distinguished by cross-beta structure. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, Type 2 diabetes, and rheumatoid arthritis. Amyloids are distinguished into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focussing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model; investigating fibril polymorphism; and relating amyloids to native conformations. In FY12 we focussed on the following projects. 1) Disposition and role of the highly charged middle domain (M-domain) of the Sup35p prion protein. In yeast cells infected with the PSI+ prion, the protein Sup35p forms aggregates and its activity in translation termination is down-regulated but not eliminated. Sup35p has an N-terminal prion domain; a highly charged M-domain of about 125 residues; and a functional C-terminal domain. By negative staining, cryo-EM, and scanning transmission EM (STEM), in vitro-assembled filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse cloud of C-domains, giving a full diameter of 65nm. In diameter (8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield 1 subunit per 0.47 nm for N-fibrils, NM-fibrils, and Sup35p filaments, further supporting the amyloid fibril backbone model. The 30nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations. The extended M-domain conformations offer an explanation for residual Sup35p activity in infected cells, whereby the C-domains remain free enough to be capable of some interaction with ribosomes (1). 2) HET-s is a prion protein of the fungus Podospora anserina. The properties of amyloid fibrils formed by its C-terminal prion domain depend on the pH of assembly; above pH 3, infectious singlet fibrils are produced, and below pH 3, non-infectious triplet fibrils. To investigate the correlation between structure and infectivity, we performed cryo-EM analyses. Singlet fibrils have an axial periodicity of 40.9 nm and a left-handed twist. Triplet fibrils have three protofibrils whose profile and dimensions (4.0 x 2.5 nm) and axial packing (1 subunit per 0.94 nm) match those of singlets but differ in their supercoiling. At 0.79 nm resolution, the cross-section of the singlet fibril reconstruction is consistent with that of a model previously determined by solid-state NMR. We identified the inter-protofibril surfaces in triplet fibrils by fitting this model into cryo-EM density maps; on this basis, we infer that stacks of salt bridges are important for inter-protofibril interactions. Finally, we discuss models of prion infectivity based on templating, conventionally assumed to take place at fibril ends. For HET-s, templating from the lateral surface that is exposed in singlets and occluded in triplets could explain their respective infectivities (2). 3) Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In the past year, we published a review (3) that specifically assesses the contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Current research is focussed primarily on optimizing the production of well ordered filaments of Ure2p-related constructs for high resolution cryo-EM analyses. Towards this goal, we have experimented with substituting different globular proteins for the native C-terminal domain of Ure2p. After a considerable amount of trial-and-error, we have identified a fusion construct with favorable properties. importantly these filaments do not aggregate under hypotonic condition at pH 8.0-8.5. Nor does it assemble rapidlyly, allowing enough time for purification and optimized polymerization. We are also advancing a project aimed at characterizing amyloid fibrils assembled from poly-Glu-rich proteins which have been implicated in a number of neurodegenerative diseases.